Candida albicans Infections: a novel porcine wound model to evaluate treatment efficacy.

Candida albicans is a common cause of opportunistic mycoses worldwide and a major contributor in wound infections. The purpose of this study was to establish a fungal wound model and analyze the effects of a common antifungal agent against the proliferation of three C. albicans strains. Second degree burns were created, and then inoculated with one of three different C. albicans ATCC strains: 10261 reference strain, 64550 fluconazole resistant and 26310 fluconazole sensitive. After fungal inoculation, every wound was covered with dressings for 4 h to allow fungal colonization on every wound bed. After 4 h, the dressings were removed, and each wound was treated either once or twice daily with a topical terbinafine hydrochloride or left untreated. On days 2, 4 and 7 post inoculation, three wounds from each treatment group were scrub cultured and quantified. On day 2, wounds infected with the sensitive strains 26310 and 10261 and treated twice showed a significant reduction when compared against those infected wounds receiving once daily treatment. On day 4, wounds which were infected with C. albicans fluconazole sensitive (ATCC 26310) showed a significant reduction in fungal cell counts with treatment applied twice daily. A significant reduction in the colony counts was exhibited in all three strains at the seventh day with active as compared to the non-treated wounds. Twice daily treatment resulted in a lower fungal count than once daily treatment. Neither treatment was able to entirely eradicate C. albicans during the duration of this study. Establishing a reliable fungal wound model will help in the translational goal of identifying new antifungal that could be used clinically by wound care providers.

Essential Oils and Supercritical CO2 Extracts of Arctic Angelica (Angelica archangelica L.), Marsh Labrador Tea (Rhododendron tomentosum) and Common Tansy (Tanacetum vulgare)-Chemical Compositions and Antimicrobial Activities.

Traditionally, arctic Finnish Angelica (Angelica archangelica L.), marsh Labrador tea (Rhododendron tomentosum, syn. Ledum palustre) and common tansy (Tanacetum vulgare) have been used as medicinal herbs in folklore medicine. However, these underutilised plants are a source of, e.g., oil-based compounds, which could benefit many modern applications implemented by the green chemistry extraction methods, as well. We extracted Angelica, marsh Labrador tea and common tansy by non-toxic and recyclable extraction methods, i.e., hydrodistillation and supercritical carbon dioxide (scCO2) extraction; characterised the essential oils (EOs) and scCO2 extracts by combination of gas chromatography and mass spectrometry (GC-MS), and in addition, analysed the antimicrobial properties. As expected for Angelica root and common tansy inflorescence, the scCO2 extraction method produced less amount of volatile compounds compared to hydrodistillation. On the other hand, more coumarins, alkanes, fatty alcohols and fatty acids were obtained. Additionally, sesquiterpenoids palustrol and ledol were predominant compounds in both marsh Labrador tea EO and scCO2 extract. According to our results, however, all the EOs and scCO2 extracts showed broad spectrum of antimicrobial activities against the selected microbes, but the effects were extract-specific. The strongest and broadest antimicrobial activities were performed by marsh Labrador tea scCO2 extract, which showed extremely strong effect on Staphylococcusaureus subsp. aureus and strong effect on Candida albicans.

Adenophora triphylla var. japonica Inhibits Candida Biofilm Formation, Increases Susceptibility to Antifungal Agents and Reduces Infection.

(1) Background: Candida is the most common cause of fungal infections worldwide, but due to the limited option of antifungal therapies, alternative strategies are required. (2) Methods: Adenophora triphylla var. japonica extract was used for the biofilm formation assay using RPMI1640. The combinatorial antifungal assay, the dimorphic transition assay, and the adherence assay were done to see the influence of inhibition of biofilm formation. qRT-PCR analysis were performed to check the gene expression. (3) Results: Adenophora triphylla var. japonica extract inhibited the Candida biofilm formation. Treatment of extract increased the antifungal susceptibility of miconazole from a 37% reduction in fungal growth to 99.05%, and also dose-dependently reduced the dimorphic transition of Candida and the attachment of Candida to HaCaT cells. The extract blocked the expression of hyphal-related genes, extracellular matrix genes, Ras1-cAMP-PKA pathway genes, Cph2-Tec1 pathway gene, and MAP kinase pathway gene. (4) Conclusions: In this study, the treatment of Adenophora triphylla var. japonica extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is a good target for the development of antifungal adjuvants, and Adenophora triphylla var. japonica extract should be a good candidate for biofilm-associated fungal infections.

The Inhibition of Non-albicans Candida Species and Uncommon Yeast Pathogens by Selected Essential Oils and Their Major Compounds.

The epidemiology of yeast infections and resistance to available antifungal drugs are rapidly increasing, and non-albicans Candida species and rare yeast species are increasingly emerging as major opportunistic pathogens. In order to identify new strategies to counter the threat of antimicrobial resistant microorganisms, essential oils (EOs) have become an important potential in the treatment of fungal infections. EOs and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities. We investigated the in vitro antifungal activity of nine commercial EOs such as Thymus vulgaris (thyme red), Origanum vulgare (oregano), Lavandula vera (lavender), Pinus sylvestris (pine), Foeniculum vulgare (fennel), Melissa officinalis (lemon balm), Salvia officinalis (sage), Eugenia caryophyllata (clove) and Pelargonium asperum (geranium), and some of their main components (α-pinene, carvacrol, citronellal, eugenol, γ-terpinene, linalool, linalylacetate, terpinen-4-ol, thymol) against non-albicans Candida strains and uncommon yeasts. The EOs were analyzed by GC-MS, and their antifungal properties were evaluated by minimum inhibitory concentration and minimum fungicidal concentration parameters, in accordance with CLSI guidelines, with some modifications for EOs. Pine exhibited strong antifungal activity against the selected non-albicans Candida isolates and uncommon yeasts. In addition, lemon balm EOs and α-pinene exhibited strong antifungal activity against the selected non-albicans Candida yeasts. Thymol inhibited the growth of all uncommon yeasts. These data showed a promising potential application of EOs as natural adjuvant for management of infections by emerging non-albicans Candida species and uncommon pathogenic yeasts.

Antivirulence activity and in vivo efficacy of a thiazole derivative against candidiasis.

Candida albicans is a pathogen equipped with a variety of commensal and virulence traits that help it colonize the microbiota and invade host tissue during infection. In this study, we investigated the potential anticandidal activity of 3-[2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazino)]butan-1-ol (MT), a thiazolylhydrazone compound synthesized by our group, and identified it as a promising antifungal agent. The activity of MT was evaluated in vitro and in vivo against C. albicans as well as its ability to inhibit virulence factors. For this, the ability of MT to inhibit the adhesion of C. albicans to human buccal epithelial cells and biofilm formation and filamentation was tested. In addition, the potential in vivo activity of MT was evaluated in murine models of oral candidiasis. Our results confirmed the antifungal activity of MT, with a minimal inhibitory concentration range of 0.5-2 µg/mL. Indeed, MT treatment in vitro decreased the expression of C. albicans genes involved in biofilm formation and morphogenesis and encoding hydrolytic enzymes, which was also confirmed through phenotypic observations. In addition, MT promoted a decrease in the colony forming units recovered from the tongues of mice with oral candidiasis. In this work, we present a potent antivirulence compound that shows potential for candidiasis therapy, especially for topical use.

The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

Paeonol alleviates dextran sodium sulfate induced colitis involving Candida albicans-associated dysbiosis.

Inflammatory bowel disease (IBD), which consists of ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammatory disorder of the gastrointestinal tract. Occurrence and development of UC have been associated with multiple potential causative factors, which include fungal dysbiosis. Growing evidence reveals that Candida albicans-associated dysbiosis is correlated with clinical deterioration in UC. Paeonol (PAE) is a commonly used traditional medicine with multiple reported properties including effective alleviation of UC. In this study, a murine UC model was established by colonizing mice with additional C. albicans via gavage prior to dextran sodium sulfate (DSS) administration. Effects of PAE treatment were also assessed at initiation and in preestablished C. albicans-associated colitis. The results showed that C. albicans supplementation could aggravate disease activity index (DAI), compromise mucosal integrity, exacerbate fecal and tissue fungal burdens, increase serum ß-glucan and anti-Saccharomyces cerevisiae antibody (ASCA) levels, promote serum and colonic tissue pro-inflammatory cytokine secretion (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8) and decrease the anti-inflammatory cytokine IL-10 level. It also stimulated Dectin-1, TLR2 and TLR4 as well as expression of their downstream effector NF-κB in colonic tissue. After PAE treatment, the adverse impacts of C. albicans on colitis were relieved, via decreased receptor-associated local and systemic inflammation. Our study suggests that PAE should be a candidate for treatment of fungal dysbiosis-associated UC and may act through the Dectin-1/NF-κB pathway in collaboration with TLR2 and TLR4. LAY SUMMARY: Candida albicans is believed to be an important stimulator in ulcerative colitice (UC) development. Suppressing the growth of intestinal C. albicans can be contributory to the amelioration of UC. Paeonol (PAE) is a commonly used traditional medicine with multiple biological functions. In this study, we observed that PAE could alleviate symptoms in mice UC model accompanying with burden reduction of C. albicans. Therefore, we suppose that PAE can be a candidate in the treatment of C. albicans-associated UC.

Distillation time effecting on the composition of Origanum floribundum essential oils and their antioxidant and antimicrobial activities.

The essential oils (EOs) of Origanum floribundum Munby, an aromatic and medicinal plant endemic in Algeria, were extracted by different hydrodistillation times (30 min, 1, 2 and 3 h) and analyzed by GC and GC-MS. The chromatographic analysis showed that thymol (32.7-45.0%), p-cymene (16.8-23.1%) and γ-terpinene (21.6-28.7%) were the most prominent components of the oils. The antioxidant ability was measured using the reductive potential, thiobarbituric acid reactive substances (TBARS) assay and the inhibition of free radicals DPPH● and ABTS●+. Antibacterial activity was assessed by the disc diffusion method against three bacteria (Escherichia coli, Staphylococcus aureus and Bacillus subtilis) and one fungus (Candida albicans). Minimal inhibitory concentrations (MICs) were determined using a microdilution method. Thymol is one of the compounds of EOs, which are widely reported as very biologically active. Although the oil isolated for 30 min was the less-thymol rich, it was the most active with all the antioxidant tests. In the most cases, the antimicrobial activity showed the best results with oils isolated for 30 min and 3 h (MIC = 0.25-1.75 µL/mL). These results suggest that it might be possible to isolate the EO from this plant for a minimum distillation time to obtain oil that can give maximum biological activities.

Antifungal and anti-inflammatory potential of the endangered aromatic plant Thymus albicans.

Thymus albicans is an endemic species of the Iberian Peninsula with a vulnerable conservation status. In an attempt to contribute to the valorization of this species, the present study brings new insights on the antifungal and anti-inflammatory mechanism of action of T. albicans essential oil. The antifungal activity of the oil and its major compounds was assessed for the first time against standard and clinically isolated strains of yeasts and filamentous fungi. The effect on the two major virulence factors of Candida albicans (germ tube formation and biofilm disruption) was considered in more detail. At 0.08 µL/mL, the oil inhibited C. albicans germ tube formation by more than 40% and decreased biofilm biomass at MIC values, thus pointing out its antivirulent potential. The anti-inflammatory activity of the essential oil was investigated on LPS-stimulated mouse macrophages (RAW 264.7) by evaluating the levels of several pro-inflammatory mediators, namely nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). T. albicans oil reduced the production of nitrites, a NO derived sub-product, at non-cytotoxic concentrations of 0.32 and 0.64 µL/mL, by 27 and 41%, respectively. In addition, the iNOS protein levels of essential oil pre-treated cells were reduced by 14%. Overall, the high essential oil yield of T. albicans as well as its bioactive effects at concentrations without cytotoxicity, encourage further studies on the potential pharmacological applications of this species. Furthermore, these results raise awareness for the need to preserve endangered species that may hold relevant medicinal value.

Preventing Candida albicans from subverting host plasminogen for invasive infection treatment.

Candida albicans is a common fungal pathogen in humans that colonizes the skin and mucosal surfaces of the majority healthy individuals. How C. albicans disseminates into the bloodstream and causes life-threatening systemic infections in immunocompromised patients remains unclear. Plasminogen system activation can degrade a variety of structural proteins in vivo and is involved in several homeostatic processes. Here, for the first time, we characterized that C. albicans could capture and "subvert" host plasminogen to invade host epithelial cell surface barriers through cell-wall localized Eno1 protein. We found that the "subverted" plasminogen system plays an important role in development of invasive infection caused by C. albicans in mice. Base on this finding, we discovered a mouse monoclonal antibody (mAb) 12D9 targeting C. albicans Eno1, with high affinity to the 254FYKDGKYDL262 motif in α-helices 6, ß-sheet 6 (H6S6) loop and direct blocking activity for C. albicans capture host plasminogen. mAb 12D9 could prevent C. albicans from invading human epithelial and endothelial cells, and displayed antifungal activity and synergistic effect with anidulafungin or fluconazole in proof-of-concept in vivo studies, suggesting that blocking the function of cell surface Eno1 was effective for controlling invasive infection caused by Candida spp. In summary, our study provides the evidence of C. albicans invading host by "subverting" plasminogen system, suggesting a potential novel treatment strategy for invasive fungal infections.

Broad-Spectrum Antimicrobial and Antibiofilm Activity of a Natural Clay Mineral from British Columbia, Canada.

Worldwide increases in antibiotic resistance and the dearth of new antibiotics have created a global crisis in the treatment of infectious diseases. These concerns highlight the pressing need for novel antimicrobial agents. Natural clay minerals have a long history of therapeutic and biomedical applications and have lately received specific attention for their potent antimicrobial properties. In particular, Kisameet clay (KC) has strong antibacterial activity against a variety of multidrug-resistant (MDR) bacterial pathogens in vitro Here, we have extended the known spectrum of activity of KC by demonstrating its efficacy against two major fungal pathogens, Candida albicans and Cryptococcus neoformans In addition, KC also exhibits potent activity against the opportunistic bacterial pathogen Mycobacterium marinum, a model organism for M. ulcerans infection. Moreover, aqueous KC leachates (KC-L) exhibited broad-spectrum antibacterial activity, eradicated Gram-negative and Gram-positive biofilms, and prevented their formation. The mechanism(s) underlying KC antibacterial activity appears to be complex. Adjusting KC-L to neutral pH rendered it inactive, indicating a contribution of pH, although low pH alone was insufficient for its antibacterial activity. Treatment of KC minerals with cation-chelating agents such as EDTA, 2,2'-bipyridyl, and deferoxamine reduced the antibacterial activity, while supplementation of KC-L with these chelating agents eliminated the inhibitory activity. Together, the data suggest a positive role for divalent and trivalent cations, including iron and aluminum, in bacterial inhibition by KC. Collectively, these studies demonstrate the range of KC bioactivity and provide a better understanding of the mechanism underlying its antibacterial effects.IMPORTANCE The escalating emergence of multidrug-resistant (MDR) bacteria, together with the paucity of novel antimicrobial agents in antibiotic development, is recognized as a worldwide public health crisis. Kisameet clay (KC), found in British Columbia (BC), Canada, is a clay mineral with a long history of therapeutic applications among people of the First Nations. We previously reported the antibacterial activity of KC against a group of MDR clinical pathogens. Here, we demonstrate its activity against two major human-pathogenic fungal species, as well as against bacterial biofilms, which underlie many recalcitrant bacterial infections. In these studies, we also identified several geochemical characteristics of KC, such as metal ions and low pH, which are involved in its antibacterial activity. These findings provide a better understanding of the components of KC antibacterial activity and a basis for developing defined preparations of this clay mineral for therapeutic applications.

Pectin based banana peel extract as a stabilizing agent in zinc oxide nanoparticles synthesis.

Various amounts of banana peel extract were successfully used as a stabilizing agent in the co-precipitation of zinc oxide nanoparticles. The obtained materials were characterized by means of Fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), X-ray diffraction (XRD), photoluminescence spectroscopy (PL), scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDS), N2 physisorption, thermogravimetric analysis (TGA), and quadrupole mass spectroscopy (QMS). On account of using such a broad spectrum of analytic methods, a thorough description of the interactions between the organic ingredients of the extract and ZnO particles was presented. It was indicated that the banana peel extract is based on pectin. These carbohydrate macromolecules adsorb on ZnO surface due to presence of active carboxylic groups. By increasing the concentration of polysaccharides, pectin-pectin interactions were also observed. The amount of the extract used for the synthesis significantly influenced the crystalline structure of zinc oxide particles along with their size and morphology. The shape and size were varying from thin flakes (450 × 24 nm) when the smallest amount of the extract was used, through nanocones with pointed tips (210 × 120 nm) agglomerated in a flower-like structure, until cubic-shaped nanoparticles (20-40 nm) agglomerated in a pinecone-like structure (430 × 180 nm) when the biggest amount of the extract was applied. The obtained particles have displayed apromising antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria, and fungus (Candida albicans). The highest activity was demonstrated against S. aureus pathogen.

Dihydrofolate Reductase Is a Valid Target for Antifungal Development in the Human Pathogen Candida albicans.

While the folate biosynthetic pathway has provided a rich source of antibacterial, antiprotozoal, and anticancer therapies, it has not yet been exploited to develop uniquely antifungal agents. Although there have been attempts to develop fungal-specific inhibitors of dihydrofolate reductase (DHFR), the protein itself has not been unequivocally validated as essential for fungal growth or virulence. The purpose of this study was to establish dihydrofolate reductase as a valid antifungal target. Using a strain with doxycycline-repressible transcription of DFR1 (PTETO-DFR1 strain), we were able to demonstrate that Dfr1p is essential for growth in vitro Furthermore, nutritional supplements of most forms of folate are not sufficient to restore growth when Dfr1p expression is suppressed or when its activity is directly inhibited by methotrexate, indicating that Candida albicans has a limited capacity to acquire or utilize exogenous sources of folate. Finally, the PTETO-DFR1 strain was rendered avirulent in a mouse model of disseminated candidiasis upon doxycycline treatment. Collectively, these results confirm the validity of targeting dihydrofolate reductase and, by inference, other enzymes in the folate biosynthetic pathway as a strategy to devise new and efficacious therapies to combat life-threatening invasive fungal infections.IMPORTANCE The folate biosynthetic pathway is a promising and understudied source for novel antifungals. Even dihydrofolate reductase (DHFR), a well-characterized and historically important drug target, has not been conclusively validated as an antifungal target. Here, we demonstrate that repression of DHFR inhibits growth of Candida albicans, a major human fungal pathogen. Methotrexate, an antifolate, also inhibits growth but through pH-dependent activity. In addition, we show that C. albicans has a limited ability to take up or utilize exogenous folates as only the addition of high concentrations of folinic acid restored growth in the presence of methotrexate. Finally, we show that repression of DHFR in a mouse model of infection was sufficient to eliminate host mortality. Our work conclusively establishes DHFR as a valid antifungal target in C. albicans.

Derivatives of the Antimalarial Drug Mefloquine Are Broad-Spectrum Antifungal Molecules with Activity against Drug-Resistant Clinical Isolates.

The antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multidrug-resistant pathogens, such as Candida auris Here, we report that derivatives of the antimalarial drug mefloquine have broad-spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of antifungal drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles obtained using human cell lines indicated that the toxicity profiles of the mefloquine derivatives are very similar to those of the parent mefloquine, despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, subinhibitory concentrations of the mefloquine derivatives inhibited the expression of virulence traits, including filamentation in Candida albicans and capsule formation/melanization in Cryptococcus neoformans Mode/mechanism-of-action experiments indicated that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multitarget mechanism of action. The broad-spectrum scope of activity, blood-brain barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the invertebrate model Caenorhabditis elegans: First mechanistic insights.

The increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols, such as probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell model and the in vivo Caenorhabditis elegans model. In Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth (~2 log CFU.mL-1) and adhesion (150 to 6,300 times less) of the pathogen. Moreover, in addition to having a pro-longevity activity in the nematode (+42.9%, p = 3.56.10-6), Lcr35® protects the animal from the fungal infection (+267% of survival, p < 2.10-16) even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signalling pathway and genes involved in the antifungal response induced by Lcr35®, suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16/FOXO transcription factor, implicated in the longevity and antipathogenic response of C. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16 but also by suppressing the virulence of the pathogen.

Candida albicans Infection Model in Zebrafish (Danio rerio) for Screening Anticandidal Drugs.

BACKGROUND: Candida albicans is an opportunistic fungal pathogen which causes systemic infections in human. In this study, C. albicans infection model was developed in zebrafish to understand the host-pathogen interactions for straightforward anticandidal drug screening. METHODS: To develop the infection, 1 × 106 cells of C. albicans suspended in phosphate-buffered saline were deposited in zebrafish dorsal muscle by manually operated syringe. The infection progression was externally assessed by a scale of wound-healing events, based on visible changes of yeast deposited in the muscle tissues. Chemotherapy was carried out with known antifungal drugs (fluconazole, nystatin, and amphotericin B) and a potential antifungal agent, chitosan silver nanocomposites (CAgNC), after the infection as direct exposure in the water. Histopathological analysis was performed to identify the pathogen virulence and the host-pathogen interaction during the infection. RESULTS: The light microscopic observations and histopathological analysis revealed the yeast-hyphae transition at the site of infection (at 72 hpi) and progression of the infection in the host tissues. The larval survival rate under fluconazole (up to 80 µg mL-1) and nystatin (up to 20 µg mL-1) was > 90% and for CAgNC it was 40% at 36 h post-exposure (hpe). The infection progression was suppressed with the fungicidal treatments. Among inflammatory genes, il-1ß has been highly upregulated (14.68-fold) at 24 h post infection (hpi). Both il-1ß and tnf-α were moderately upregulated in infected fish gills at 72 hpi. Among the C. albicans antioxidant genes, cat1 and sod2 have been upregulated during the infection, and relative expression folds were increased from low to moderate levels with the time. DISCUSSION: We demonstrate the approach for the development of artificial infection model of zebrafish with C. albicans. By this mini vertebrate zebrafish model, researchers will be able to study novel anticandidal compounds in vivo with respect to the host, pathogen, and their interactions.

Tinospora cordifolia Aqueous Extract Alleviates Cyclophosphamide- Induced Immune Suppression, Toxicity and Systemic Candidiasis in Immunosuppressed Mice: In vivo Study in Comparison to Antifungal Drug Fluconazole.

OBJECTIVE: The present study was aimed to evaluate the effect of the aqueous extract of Tinospora cordifolia (AETC) against cyclophosphamide-induced immunosuppression and systemic Candida albicans infection in a murine model. METHODS: The protective effect of AETC against cyclophosphamide-induced leukopenia was evaluated by quantitative and qualitative analysis of the leukocytes. The immune-stimulating potential of AETC on macrophages was assessed by determining the levels of secreted cytokines. To determine the direct antifungal activity, AETC or fluconazole was administered to C. albicans infected mice. The efficacy of treatment was assessed by determining the survival rate, kidney fungal burden, the organ index and liver inflammation parameters. RESULTS: Cyclophosphamide administration resulted in substantial depletion of leukocytes, whereas AETC treatment induced the recovery of leukocytes in cyclophosphamide-injected mice. Moreover, AETC treatment of macrophages resulted in enhanced secretion of IFN-γ, TNF-α and IL-1ß. C. albicans infected mice treated with AETC at the doses of 50 and 100 mg/kg exhibited 40% and 60% survival rate, whereas the mice treated with fluconazole at a dose of 50 mg/kg showed 20% survival rate. Like survival data, the fungal load was found to be the lowest in the kidney tissues of mice treated with AETC at a dose of 100 mg/kg. Interestingly, mice infected with C. albicans demonstrated improvement in the organ indices and liver functioning after AETC treatment. CONCLUSION: These results suggest that AETC may potentially be used to rejuvenate the weakened immune system and eliminate systemic candidiasis in mice.

Drosophila melanogaster as a model to study virulence and azole treatment of the emerging pathogen Candida auris.

OBJECTIVES: Candida auris is an emerging, often MDR, yeast pathogen. Efficient animal models are needed to study its pathogenicity and treatment. Therefore, we developed a C. auris fruit fly infection model. METHODS: TollI-RXA/Tollr632 female flies were infected with 10 different C. auris strains from the CDC Antimicrobial Resistance bank panel. We used three clinical Candida albicans strains as controls. For drug protection assays, fly survival was assessed along with measurement of fungal burden (cfu/g tissue) and histopathology in C. auris-infected flies fed with fluconazole- or posaconazole-containing food. RESULTS: Despite slower in vitro growth, all 10 C. auris isolates caused significantly greater mortality than C. albicans in infected flies, with >80% of C. auris-infected flies dying by day 7 post-infection (versus 67% with C. albicans, P < 0.001-0.005). Comparison of C. auris isolates from different geographical clades revealed more rapid in vitro growth of South American isolates and greater virulence in infected flies, whereas the aggregative capacity of C. auris strains had minimal impact on their growth and pathogenicity. Survival protection and decreased fungal burden of fluconazole- or posaconazole-fed flies infected with two C. auris strains were in line with the isolates' disparate in vitro azole susceptibility. High reproducibility of survival curves for both non-treated and antifungal-treated infected flies was seen, with coefficients of variation of 0.00-0.31 for 7 day mortality. CONCLUSIONS: Toll-deficient flies could provide a fast, reliable and inexpensive model to study pathogenesis and drug activity in C. auris candidiasis.

Effect of Trigonella foenum Extract and ZiO2 Nanoparticles on Some Pathogenic Fungi and Bacteria

Aim: The current venture, were made to evaluate the inhibitory effect of Trigonella foenum seed Extract and ZiO2 Nanoparticles on some selected species of Fungi and Bacteria. Materials and Methods: two bacterial species included Pseudomonas aeruginosa and Staphylococcus aureus and three fungal species which is Cryptococcus neoformans, Candidda albicans and Chaetomium were used to evaluate the antibacterial activity of Trigonella foenum Extract and ZiO2 Nanoparticles. Results: This study showed that the Zirconium Oxide (ZiO2) nanoparticles have antifungal and antibacterial activities on the isolates of Cryptococcus neoformans, Candida alicans and Staphylococcus aureus, respectively. While the antimicrobial activity of Zirconium Oxide nanoparticles on the Chaetomium and Pseudomonas aeruginosa was negative. All tested fungi and bacterial isolates were found to be sensitive to Trigonella foenum seed extract, the results of the compination of the ZiO2 Nanoparticle and the Trigonella foenum seed extract were poisitive for all tested fungi isolates and bacterial isolates. The XRD analysis was done for Zirconium Oxide nanoparticles and the result showed that the biocrystallization on the surface of the Zirconium Oxide manoparticles. The average partides size was about (29.8) nm. Conclusions: This investigation conclude that the use of Trigonella foenum seed Extract has the effect of killing all bacteria and fungi under study, result indicate the Trigonella foenun seed Extract best antibacterial efficacy than the ZiO2 together (AU)

Repurposing of Fluvastatin Against Candida albicans CYP450 Lanosterol 14 α-demethylase, a Target Enzyme for Antifungal Therapy: An In silico and In vitro Study.

BACKGROUND: The incidence of fungal infections has increased significantly. Specifically the cases of candida albicans infection are increasing day by day and their resistance to clinically approved drugs is a major concern for humans. Various classes of antifungal drugs are available in the market for the treatment of these infections but unfortunately, none of them is able to treat the infection. OBJECTIVES: Thus, in the present investigation, we have repurposed the well-known drug (Fluvastatin) in the treatment of Candida albicans infections by using in silico, in vitro and ex vivo techniques. MATERIAL AND METHODS: Computational and in vitro techniques. RESULTS: Firstly, we developed and validated a simple model of CYP45014α-lanosterol demethylase of Candida albicans by using crystal structure of Mycobacterium tuberculosis (1EA1). Further, fluvastatin was docked with a validated model of CYP45014α-lanosterol demethylase and revealed good binding affinity as that of fluconazole. In vitro results (Percentage growth retardation, Fungal growth kinetics, Biofilm test and Post antifungal test) have shown good antifungal activity of fluvastatin. Finally, the results of MTT assay have shown non-cytotoxic effect of fluvastatin in murine splenocytes and thymocytes. CONCLUSION: However, further in vivo studies are required to confirm the complete role of fluvastatin as an antifungal agent.